This project is designed to study several aspects of human megakaryocytopoiesis. We will exploit a newly developed, semi-solid clonal assay system for the human megakaryocyte colony-forming progenitor cell or CFU-M. The physiology of normal human megakaryocytopoiesis will be investigated by studying the physical characteristics, cell cycle, and surface markers of the human CFU-M. Regulatory influences including circulating hormones, iron, and cellular interactions will be evaluated. A significant effort will be directed towards the study of a newly described circulating substance (present in sera of patients with aplastic anemia) which markedly augments in vitro CFU-M colony formation. This substance, called megakaryocyte colony stimulating activity or Meg-CSA, will be purified and characterized using a variety of protein chemistry techniques. Its mechanism of action on the CFU-M and possible sites of production will be elucidated. Other studies will focus on the alterations in megakaryocytopoiesis present in a variety of human diseases. Both circulating levels of Meg-CSA and the number and characteristics of bone marrow and peripheral blood CFU-M will examined. Serial Meg-CSA levels will be assayed in patients receiving cytotoxic chemotherapy. Mechanisms of amegakaryocytic thrombocytopenia will be investigated using lymphocyte depletion/addition and mixing experiments, and preincubations with antibody and complement. Conversely, the cause of reactive thrombocytosis will be studied by examining quantitatively and qualitatively the CFU-M in these patients and evaluation for instances of autonomous Meg-CSA production. These proposed studies are unique in their human focus and in their ability to examine the early steps of platelet production, i.e. the steps from stem cell to megakaryocyte.